1. Department of Chemistry, University of California, Berkeley, California 94720, USA
2. Department of Molecular, Cell, and Developmental Biology, and Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA
3. Biophysics Graduate Group, University of California, Berkeley, California 94720, USA
4. Brazilian Synchrotron Light Laboratory, Caixa Postal 6192, Campinas SP 13083-970, Brazil
5. Graduate School of Biostudies, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto, 606-8501, Japan
6. Howard Hughes Medical Institute, Department of Physics and Molecular and Cell Biology, University of California, Berkeley, California 94720, USA

Correspondence to: Ignacio Tinoco¹ Correspondence and requests for materials should be addressed to I.T. (Email: intinoco@lbl.gov).

Abstract

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation-and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases—one codon—and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.

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